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1.
The EF‐1α promoter maintains high‐level transgene expression from episomal vectors in transfected CHO‐K1 cells 下载免费PDF全文
Xi Zhang Danhua Xu Qin Li Junhe Zhang Tianyun Wang 《Journal of cellular and molecular medicine》2017,21(11):3044-3054
In our previous study, we demonstrated that episomal vectors based on the characteristic sequence of matrix attachment regions (MARs) and containing the cytomegalovirus (CMV) promoter allow transgenes to be maintained episomally in Chinese hamster ovary (CHO) cells. However, the transgene expression was unstable and the number of copies was low. In this study, we focused on enhancers, various promoters and promoter variants that could improve the transgene expression stability, expression magnitude (level) and the copy number of a MAR‐based episomal vector in CHO‐K1 cells. In comparison with the CMV promoter, the eukaryotic translation elongation factor 1 α (EF‐1α, gene symbol EEF1A1) promoter increased the transfection efficiency, the transgene expression, the proportion of expression‐positive clones and the copy number of the episomal vector in long‐term culture. By contrast, no significant positive effects were observed with an enhancer, CMV promoter variants or CAG promoter in the episomal vector in long‐term culture. Moreover, the high‐expression clones harbouring the EF‐1α promoter tended to be more stable in long‐term culture, even in the absence of selection pressure. According to these findings, we concluded that the EF‐1α promoter is a potent regulatory sequence for episomal vectors because it maintains high transgene expression, transgene stability and copy number. These results provide valuable information on improvement of transgene stability and the copy number of episomal vectors. 相似文献
2.
Reagent storage has been a long-standing challenge for diagnostics, especially those designed for low-resource settings and point-of-care applications. In general, the stability of a reagent relies on careful temperature control, often by refrigeration, which is costly and often unavailable in these remote settings. Poor reagent integrity can negatively affect the reproducibility and reliability of an assay. Given the recent interest in paper-based devices designed for quantitative analysis in point-of-care settings, a better understanding of reagent stability on filter paper is critical for proper device use and its longevity. In this article, we present an independent method to examine the stability of reconstituted antibodies that were stored on filter paper using flow cytometry. We validated the method by measuring the activity as measured by the mean fluorescence intensity (MFI) of antibodies stored with known stabilizers. Furthermore, we demonstrated the potential of our method to screen the influence of other paper treatments and storage processes on antibody stability, which may be applicable to the storage of reagents on paper in general. 相似文献
3.
《DNA Repair》2017
Infliction of DNA damage initiates a complex cellular reaction – the DNA damage response – that involves both signaling and DNA repair networks with many redundancies and parallel pathways. Here, we reveal the three strategies that the simple multicellular eukaryote, C. elegans, uses to deal with DNA damage induced by light. Separately inactivating repair or replicative bypass of photo-lesions results in cellular hypersensitivity towards UV-light, but impeding repair of replication associated DNA breaks does not. Yet, we observe an unprecedented synergistic relationship when these pathways are inactivated in combination. C. elegans mutants that lack nucleotide excision repair (NER), translesion synthesis (TLS) and alternative end joining (altEJ) grow undisturbed in the dark, but become sterile when grown in light. Even exposure to very low levels of normal daylight impedes animal growth. We show that NER and TLS operate to suppress the formation of lethal DNA breaks that require polymerase theta-mediated end joining (TMEJ) for their repair. Our data testifies to the enormous genotoxicity of light and to the demand of multiple layers of protection against an environmental threat that is so common. 相似文献
4.
Identifying the contributions to thermodynamic stability of capsids is of fundamental and practical importance. Here we use simulation to assess how mutations affect the stability of lumazine synthase from the hyperthermophile Aquifex aeolicus, a T = 1 icosahedral capsid; in the simulations the icosahedral symmetry of the capsid is preserved by simulating a single pentamer and imposing crystal symmetry, in effect simulating an infinite cubic lattice of icosahedral capsids. The stability is assessed by estimating the free energy of association using an empirical method previously proposed to identify biological units in crystal structures. We investigate the effect on capsid formation of seven mutations, for which it has been experimentally assessed whether they disrupt capsid formation or not. With one exception, our approach predicts the effect of the mutations on the capsid stability. The method allows the identification of interaction networks, which drive capsid assembly, and highlights the plasticity of the interfaces between subunits in the capsid. Proteins 2015; 83:1733–1741. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc 相似文献
5.
The impact of high‐order epistasis in the within‐host fitness of a positive‐sense plant RNA virus 下载免费PDF全文
RNA viruses are the main source of emerging infectious diseases because of the evolutionary potential bestowed by their fast replication, large population sizes and high mutation and recombination rates. However, an equally important property, which is usually neglected, is the topography of the fitness landscape. How many fitness maxima exist and how well they are connected is especially interesting, as this determines the number of accessible evolutionary pathways. To address this question, we have reconstructed a region of the fitness landscape of tobacco etch potyvirus constituted by mutations observed during the experimental adaptation of the virus to the novel host Arabidopsis thaliana. Fitness was measured for many genotypes and showed the existence of multiple peaks and holes in the landscape. We found prevailing epistatic effects between mutations, with cases of reciprocal sign epistasis being common among pairs of mutations. We also found that high‐order epistasis was as important as pairwise epistasis in their contribution to fitness. Therefore, results suggest that the landscape was rugged due to the existence of holes caused by lethal genotypes, that a very limited number of potential neutral paths exist and that it contained a single adaptive peak. 相似文献
6.
8.
Vegetation,fire and soil feedbacks of dynamic boundaries between rainforest,savanna and grassland 下载免费PDF全文
Harry J. MacDermott Roderick J. Fensham Quan Hua David M. J. S. Bowman 《Austral ecology》2017,42(2):154-164
At fine spatial scales, savanna‐rainforest‐grassland boundary dynamics are thought to be mediated by the interplay between fire, vegetation and soil feedbacks. These processes were investigated by quantifying tree species composition, the light environment, quantities and flammability of fuels, bark thickness, and soil conditions across stable and dynamic rainforest boundaries that adjoin grassland and eucalypt savanna in the highlands of the Bunya Mountains, southeast Queensland, Australia. The size class distribution of savanna and rainforest stems was indicative of the encroachment of rainforest species into savanna and grassland. Increasing dominance of rainforest trees corresponds to an increase in woody canopy cover, the dominance of litter fuels (woody debris and leaf), and decline in grass occurrence. There is marked difference in litter and grass fuel flammability and this result is largely an influence of strongly dissimilar fuel bulk densities. Relative bark thickness, a measure of stem fire resistance, was found to be generally greater in savanna species when compared to that of rainforest species, with notable exceptions being the conifers Araucaria bidwillii and Araucaria cunninghamii. A transect study of soil nutrients across one dynamic rainforest – grassland boundary indicated the mass of carbon and nitrogen, but not phosphorus, increased across the successional gradient. Soil carbon turnover time is shortest in stable rainforest, intermediate in dynamic rainforest and longest in grassland highlighting nutrient cycling differentiation. We conclude that the general absence of fire in the Bunya Mountains, due to a divergence from traditional Aboriginal burning practices, has allowed for the encroachment of fire‐sensitive rainforest species into the flammable biomes of this landscape. Rainforest invasion is likely to have reduced fire risk via changes to fuel composition and microclimatic conditions, and this feedback will be reinforced by altered nutrient cycling. The mechanics of the feedbacks here identified are discussed in terms of landscape change theory. 相似文献
9.
城市森林景观格局与过程研究进展 总被引:14,自引:1,他引:13
大规模的城市化进程,改变了城市以及周边区域的景观格局,显著影响着城市森林生态系统的结构、过程与功能.景观尺度上的城市森林景观格局与生态过程研究已经成为当前城市森林学的研究热点.在阐明城市森林及其景观格局与过程概念的基础上,综述了国内外城市森林景观格局与过程的研究内容、研究方法的进展情况,指出了以下几个方面有望成为今后城市森林景观研究的发展方向:景观尺度上的城市森林系统能流、物质循环等生态学过程研究;城市森林景观格局演变的社会驱动力研究;基于生态服务功能的城市森林规划研究;半都市化地区的森林景观格局与过程研究;基于空间显式景观模型的城市森林景观格局与过程模块开发. 相似文献
10.
Solar Cells: Role of Microstructure in Oxygen Induced Photodegradation of Methylammonium Lead Triiodide Perovskite Films (Adv. Energy Mater. 20/2017) 下载免费PDF全文